The long-term goal of this research is to determine the mechanism by which mechanical loading leads to bone formation. Mechanical stimulation is critical for the homeostasis of extracellular matrix in connective tissues such as bone and is responsible for determining the bone architecture. These effects on bone are dependent on the magnitude, duration, and rate of the mechanical load stimuli. There is evidence that the anabolic effects of bone vary with strain magnitude and strain rates. The anabolic factors released by mechanical stimuli are potential candidates for use as therapeutic agents to promote fracture healing, to incorporate bone into implant surfaces and bone graft materials, and to increase bone formation in osteoporosis. The response to mechanical stimuli by the stimulated cells is likely to involve the activation of specific signaling pathways. One pathway that has been shown to be involved in response to mechanical stimuli is the protein kinase C (PKC) pathway, which our laboratory has been investigating in bone. This study will focus on defining the strain rates and magnitudes which elicit the maximum anabolic bone response, identification of the cells responding and growth factors released in response to mechanical stimuli, and on the role of the PKC pathway in the actions of mechanical stimulation. This study uses a micromechanical-testing device capable of delivering both cyclic and/or step load in milli-Newton loads and micrometer displacements to viable neonatal mouse tibial bone. We will apply different loading regimens to bone tissue and assess the anabolic response and the differential expression of the local regulators of bone formation. We will assess the effect of specific PKC inhibitors and antisense oligonucleotides to specific PKC isozymes and determine the changes in bone anabolic response to mechanical stimuli. The hypothesis is that a) bone formation is maximized under certain conditions of strain rate and magnitude, b) the cells responding to the mechanical stimuli are the osteocytes, which release local bone regulators to mediate bone formation, and c) this effect involves specific PKC isozymes. The Specific Aims of this proposal are; (1) to assess the effect of load magnitude and frequency on bone formation response; (2) to identify the cells responding to the mechanical stimuli and determine the differential expression of local regulators of bone formation, BMP2, BMP7, TGFbeta, IGF1, and IGF2; and (3) to characterize the role of the PKC signal transduction pathway in the anabolic response of bone to mechanical stimuli.